Anti-TFPI antibody variants with differential binding across PH range for improved pharmacokinetics

ABSTRACT

Antibodies are disclosed that bind to and inhibit the anti-coagulant function of TFPI and have a lower affinity for TFPI at pH 6.0 than at pH 7.4. The lower affinity at pH 6 improves circulating half-life (T½) due to reduced target mediated clearance, a process by which an antibody/antigen complex is endocytosed and trafficked to the lysosome where both components are degraded. The lower affinity at pH 6.0 results in disruption of the complex prior to lysosome targeting and allows for re-circulation of the antibody. Specific modifications to antibody binding by histidine residue substitution are disclosed along with methods of use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/798,261 filed Mar. 15, 2013, which application is incorporated hereinby reference.

BACKGROUND

Currently the prophylactic management of hemophilia A and B patients isreplacement of either FVIII or FIX (recombinant or plasma-derivedproducts). These treatments are administered two or three times perweek, placing a heavy burden on patients to comply with theirprophylactic regime. Despite rigorous management and strict adherence,patients typically experience occasional breakthrough bleeds and requireon-demand treatments. If not managed properly, frequent and severebleeding leads to significant morbidities, especially hemarthropathy.Despite the proven efficacy of the existing agents used to treathemophilia A and B patients, most adolescents, teenagers and olderadults decide to lessen the burden of prophylaxis by reducing the numberof injections taken on a routine basis. This approach furthercompromises the protection needed to adequately manage bleeds.

Consequently, an agent that significantly provides protection andrequires relatively infrequent administration is most desired. Theoptimal therapy should provide protection via weekly or less frequentdosing. Given the current competitive environment, once a week therapiesadministered intravenously (i.v.) or subcutaneously (s.c.) may be areality over the next 3-4 years. Therefore, an agent that isadministered i.v. or s.c. should provide a superior administrationprofile with commensurate protection. In the event subcutaneousadministration can be achieved, once a week dosing could also offer asignificant value to the future treatment landscape due to the reducedinvasiveness of this approach.

Another major issue for current hemophilia therapy is the development ofinhibitory antibodies against Factor VIII or Factor IX. Approximately25% of FVIII treated patients generate inhibitors, or neutralizingantibodies against FVIII. Inhibitors are also found in FIX treatedpatients, although less frequently. The development of inhibitorssignificantly reduces the effectiveness of replacement therapy andprovides a challenge for managing bleeds in hemophilia patients. Thecurrent treatment for bleeds in patients with inhibitors to FVIII or FIXis bypass therapy with recombinant Factor VIIa or plasma derived FEIBA.The half-life of rFVIIa is quite short (−2 hours) and thus prophylactictreatment in these patients is uncommon [Blanchette, Haemophilia 16(supplement 3): 46-51, 2010].

To address these unmet medical needs, antibodies against Tissue FactorPathway Inhibitor (TFPI) as long-acting agents have been developed. SeeWO 2010/017196; WO 2011/109452; WO 2012/135671. TFPI is the majorinhibitor of the tissue factor initiated coagulation pathway, which isintact in Persons with Hemophilia (PWH), and thus inhibition of TFPI mayrestore hemostasis in PWH exhibiting inhibitory antibodies to FVIII orFIX. In addition to allowing access to this target, monoclonal antibody(mAb) therapeutics have been shown to have significantly longercirculating half-lives (up to 3 weeks) than recombinant replacementfactors. Antibodies that inhibit TFPI also have significantbioavailability following subcutaneous injection. Thus, anti-TFPImonoclonal antibody therapy would meet an important unmet medical needfor subcutaneous, long acting hemostatic protection for PWH and PWH withinhibitors.

However, while inhibition of TFPI has been shown to promote coagulationin hemophilic plasmas and hemophilic animals, antibodies against TFPIhave relatively short, non-linear half-lives due to a phenomenon knownas target mediated drug disposition (TMDD) a process by which theantibody is removed from circulation due to its interaction with arapidly cleared target or by being sequestered from the plasma due toits co-localization with its target, of the antibody:antigen complex.Therefore, antibodies that avoid TFPI mediated TMDD and have a prolongedhalf-life would lead to less frequent dosing and reduce the amount ofmaterial needed per dose. Furthermore, the need for a lower dose mayalso make feasible subcutaneous dosing where the dosing volume becomes alimiting step. For example, an optimized anti-TFPI antibody 2A8-g200 (WO2011/109452), has a half-life of 28 hours when dosed at 5 mg/kg and 67hrs when dosed at 20 mg/kg in non-human primates.

This relatively short half-life, and the need for larger doses toovercome TMDD, increases the injection burden on patients, limitsformulation for subcutaneous dosing and increases the costs of goods.Pharmocokinetic analysis of these antibodies in non-human primatesdemonstrates that the circulating half-life is not linear with dose,and, particularly at lower doses, is shorter than is characteristic ofantibody drugs. A similar pharmacokinetic profile was described inUS2011/0318356 A1 for another anti-TFPI antibody. This differential,with a marked shortening of T½ at lower doses, is characteristic ofTMDD, where the slower clearance at higher doses is due to saturation ofthe target.

Accordingly, an unmet medical need exists for better prophylactictreatment for moderate-to-severe hemophilia A and B, especially forthose patients with inhibitors against FVIII or FIX. This need would bemet by an anti-TFPI antibody having improved characteristics that may beadministered intravenously or subcutaneously, and at a reducedfrequency, preferably once weekly or less.

SUMMARY OF INVENTION

To increase the half-life of an anti-TFPI antibody and to reduce theinjection burden, a longer acting anti-TFPI antibody was producedwithout a loss of efficacy and tested to confirm improved properties ascompared to other anti-TFPI antibodies having demonstrated TFPI bindingcharacteristics and proven efficacy in treating coagulationdeficiencies. (See WO 2011/109452.) Specifically, TMDD is reduced bycreating a variant anti-TFPI antibody having reduced affinity at pH 6.0relative to pH 7.4. An anti-TFPI antibody may be taken into cells incomplex with its target, TFPI, through receptors involved in TFPIclearance. One receptor, identified by Narita et al. (JBC 270 (42):24800-4, 1995) is LRP (LDL receptor related protein), which targets TFPIfor degradation in the endosome. However, if this antigen:antibodycomplex can be disrupted at low pH, which is characteristic of theendosome, the antibody can be recycled via FcRn binding, therebyincreasing exposure in circulation. This principal has been shown for anantibody to PSCK9 by Chapparo-Riggers et al. JBC 287 (14): 110-7 (2012).

One method for disruption of an antigen:antibody complex at lower pH isto substitute histidine residues near the antibody:antigen interactionsurface. The amino acid histidine (His) is protonated at low pH, near pH6.0, and thus, a residue that is neutral at pH 7.4 acquires a positivecharge at pH 6.0. This can lead to charge repulsion with other aminoacids and a desirable degree of disruption or destabilization at theantibody:antigen interface.

To identify pH sensitive residues, the CDR amino acids and other aminoacids involved in antigen:antibody binding of anti-TFPI antibodies (e.g.2A8-g200) to TFPI antigen were changed individually to His. Theindividual His variants demonstrate differential binding at pH 7.4 vs.pH 6.0, and combinations of variants with differential binding have beentested for optimization.

Upon endosomal release, these pH sensitive anti-TFPI mAb variants bindto the neonatal FcRN receptor and are recycled to the plasma. Thus, acombination between a pH sensitive TFPI-binding site and a Fc domainwith increased affinity for FcRN at low pH would have a synergisticeffect that increases half-life, reduces the injection burden topatients, and reduces the cost of goods.

DESCRIPTION OF THE FIGURES

FIG. 1 shows alignment of amino acid sequences for 2A8-g200 and mutatedanti-TFPI mAbs suitable for histidine substitution (SEQ ID NOs for thesesequences can be found in Table 1). A. Variable Heavy Chain, B. VariableLight Chain. CDR regions 1-3 are indicated.

FIG. 2 shows synthesis and subcloning of a 2A8-g200 Fab HistidineScanning Library. The CDR1-3 regions are indicated with a dashed line.Underlined amino acid residues indicate the position of contact residuesto TFPI. An asterisk (*) indicates a proposed His mutation site. A).2A8-g200 heavy chain; B) 2A8-g200 light chain; C). 4B7-gB9.7 heavychain; and D) 4B7-gB9.7 light chain. Each of the SEQ. ID NOS shown inFIGS. 2A-2D can be associated with each known variant by referring toTABLE 1.

FIG. 3 shows dissociation constants at two pHs for the improvedantibodies with exemplary histidine mutations: A. L-L27H and B. L-Y31H.Surface plasmon resonance (Biacore T200) was used to measure thedissociation rate of the antibodies.

FIG. 4 shows pK profiles observed in Hem A mouse plasma over time forseveral monoclonal antibodies at concentrations of 2 mg/kg: 2A8-g200 (--

- -), histidine substituted monoclonal antibodies TPP2256 (L-Y31H/Y49H)(-^(•)-

-^(•)-) and TPP2259 (L-Y31H) (-

-). Pharmacokinetic parameters of the antibodies were determined afterintravenous (i.v.) bolus administration to HemA mouse at 2 mg/kg.

DETAILED DESCRIPTION

The term “tissue factor pathway inhibitor” or “TFPI” as used hereinrefers to any variant, isoform and species homolog of human TFPI that isnaturally expressed by cells. In a preferred embodiment of theinvention, the binding of an antibody of the invention to TFPI reducesthe blood clotting time.

As used herein, an “antibody” refers to a whole antibody and any antigenbinding fragment (i.e., “antigen-binding portion”) or single chainthereof. The term includes a full-length immunoglobulin molecule (e.g.,an IgG antibody) that is naturally occurring or formed by normalimmunoglobulin gene fragment recombinatorial processes, or animmunologically active portion of an immunoglobulin molecule, such as anantibody fragment, that retains the specific binding activity.Regardless of structure, an antibody fragment binds with the sameantigen that is recognized by the full-length antibody. For example, ananti-TFPI monoclonal antibody fragment binds to an epitope of TFPI. Theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H1) domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546), which consists of a V_(H) domain; (vi) an isolatedcomplementarity determining region (CDR); (vii) minibodies, diaboidies,triabodies, tetrabodies, and kappa bodies (see, e.g. Ill et al., ProteinEng 1997; 10:949-57); (viii) camel IgG; and (ix) IgNAR. Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are analyzed forutility in the same manner as are intact antibodies.

Furthermore, it is contemplated that an antigen binding fragment may beencompassed in an antibody mimetic. The term “antibody mimetic” or“mimetic” as used herein is meant a protein that exhibits bindingsimilar to an antibody but is a smaller alternative antibody or anon-antibody protein. Such antibody mimetic may be comprised in ascaffold. The term “scaffold” refers to a polypeptide platform for theengineering of new products with tailored functions and characteristics.

As used herein, the terms “inhibits binding” and “blocks binding” (e.g.,referring to inhibition/blocking of binding of TFPI ligand to TFPI) areused interchangeably and encompass both partial and complete inhibitionor blocking. Inhibition and blocking are also intended to include anymeasurable decrease in the binding affinity of TFPI to a physiologicalsubstrate when in contact with an anti-TFPI antibody as compared to TFPInot in contact with an anti-TFPI antibody, e.g., the blocking of theinteraction of TFPI with factor Xa or blocking the interaction of aTFPI-factor Xa complex with tissue factor, factor VIIa or the complex oftissue factor/factor VIIa by at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

Therapeutic antibodies have been made through hybridoma technologydescribed by Koehler and Milstein in “Continuous Cultures of Fused CellsSecreting Antibody of Predefined Specificity,” Nature 256, 495-497(1975). Fully human antibodies may also be made recombinantly inprokaryotes and eukaryotes. Recombinant production of an antibody in ahost cell rather than hybridoma production is preferred for atherapeutic antibody. Recombinant production has the advantages ofgreater product consistency, likely higher production level, and acontrolled manufacture that minimizes or eliminates the presence ofanimal-derived proteins. For these reasons, it is desirable to have arecombinantly produced monoclonal anti-TFPI antibody. The terms“monoclonal antibody” or “monoclonal antibody composition” as usedherein refer to a preparation of antibody molecules of single molecularcomposition. A monoclonal antibody composition displays a single bindingspecificity and affinity for a particular epitope. Generally,therapeutic antibodies for human diseases have been generated usinggenetic engineering to create murine, chimeric, humanized or fully humanantibodies. Murine monoclonal antibodies were shown to have limited useas therapeutic agents because of a short serum half-life, an inabilityto trigger human effector functions, and the production of humananti-mouse-antibodies (Brekke and Sandlie, “Therapeutic Antibodies forHuman Diseases at the Dawn of the Twenty-first Century,” Nature 2, 53,52-62, January 2003). Chimeric antibodies have been shown to give riseto human anti-chimeric antibody responses. Humanized antibodies furtherminimize the mouse component of antibodies. However, a fully humanantibody avoids the immunogenicity associated with murine elementscompletely. Thus, there is a need to develop antibodies that are humanor humanized to a degree necessary to avoid the immunogenicityassociated with other forms of genetically engineered monoclonalantibodies. In particular, chronic prophylactic treatment such as wouldbe required for hemophilia treatment with an anti-TFPI monoclonalantibody has a high risk of development of an immune response to thetherapy if an antibody with a murine component or murine origin is useddue to the frequent dosing required and the long duration of therapy.For example, antibody therapy for hemophilia A may require weekly dosingfor the lifetime of a patient. This would be a continual challenge tothe immune system. Thus, the need exists for a fully human antibody forantibody therapy for hemophilia and related genetic and acquireddeficiencies or defects in coagulation. Accordingly, the term “humanmonoclonal antibody” refers to antibodies displaying a single bindingspecificity which have at least portions of variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). These human antibodies include chimeric antibodies, such asmouse/human and humanized antibodies that retain non-human sequences.

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody that bindsto TFPI is substantially free of antibodies that bind antigens otherthan TFPI). An isolated antibody that binds to an epitope, isoform orvariant of human TFPI may, however, have cross-reactivity to otherrelated antigens, e.g., from other species (e.g., TFPI specieshomologs). Moreover, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with an affinity ofat least about 10⁵ and binds to the predetermined antigen with anaffinity that is higher, for example at least two-fold greater, than itsaffinity for binding to an irrelevant antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen. The phrases“an antibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen.”

As used herein, the term “high affinity” for an IgG antibody refers to abinding affinity of at least about 10⁷, in some embodiments at leastabout 10⁸, in some embodiments at least about 10⁹, 10¹⁰, 10¹¹ orgreater, e.g., up to 10¹³ or greater. However, “high affinity” bindingcan vary for other antibody isotypes. For example, “high affinity”binding for an IgM isotype refers to a binding affinity of at leastabout 1.0×10⁷. As used herein, “isotype” refers to the antibody class(e.g., IgM or IgG1) that is encoded by heavy chain constant regiongenes.

“Complementarity-determining region” or “CDR” refers to one of threehypervariable regions within the variable region of the heavy chain orthe variable region of the light chain of an antibody molecule that formthe N-terminal antigen-binding surface that is complementary to thethree-dimensional structure of the bound antigen. Proceeding from theN-terminus of a heavy or light chain, these complementarity-determiningregions are denoted as “CDR1,” “CDR2,” and “CDR3,” respectively. CDRsare involved in antigen-antibody binding, and the CDR3 comprises aunique region specific for antigen-antibody binding. An antigen-bindingsite, therefore, may include six CDRs, comprising the CDR regions fromeach of a heavy and a light chain V region.

As used herein, except with respect to the individual or plurality ofhistidine substitutions described below, “conservative substitutions”refers to modifications of a polypeptide that involve the substitutionof one or more amino acids for amino acids having similar biochemicalproperties that do not result in loss of a biological or biochemicalfunction of the polypeptide. A “conservative amino acid substitution” isone in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine), and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). It is envisioned that theantibodies of the present invention may have conservative amino acidsubstitutions and still retain activity.

The term “substantial homology” indicates that two polypeptides, ordesignated sequences thereof, when optimally aligned and compared, areidentical, with appropriate amino acid insertions or deletions, in atleast about 80% of amino acids, usually at least about 85%, preferablyabout 90%, 91%, 92%, 93%, 94%, or 95%, more preferably at least about96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% of the aminoacids. The invention includes polypeptide sequences having substantialhomology to the specific amino acid sequences recited herein.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, such as the AlignX™ moduleof VectorNTI™ (Invitrogen Corp., Carlsbad, Calif.). For AlignX™, thedefault parameters of multiple alignment are: gap opening penalty: 10;gap extension penalty: 0.05; gap separation penalty range: 8; % identityfor alignment delay: 40.

Another method for determining the best overall match between a querysequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe CLUSTALW computer program (Thompson et al., Nucleic Acids Research,1994, 2(22): 4673-4680), which is based on the algorithm of Higgins etal., (Computer Applications in the Biosciences (CABIOS), 1992, 8(2):189-191). In a sequence alignment the query and subject sequences areboth DNA sequences. The result of said global sequence alignment is inpercent identity. Preferred parameters used in a CLUSTALW alignment ofDNA sequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty=10, Gap Extension Penalty=0.1. For multiple alignments, thefollowing CLUSTALW parameters are preferred: Gap Opening Penalty=10, GapExtension Parameter=0.05; Gap Separation Penalty Range=8; % Identity forAlignment Delay=40.

Also provided are pharmaceutical compositions comprising therapeuticallyeffective amounts of anti-TFPI monoclonal antibody and apharmaceutically acceptable carrier. As used herein, “therapeuticallyeffective amount” means an amount of an anti-TFPI monoclonal antibodyvariant or of a combination of such antibody and factor VIII or factorIX that is needed to effectively increase the clotting time in vivo orotherwise cause a measurable benefit in vivo to a patient in need. Theprecise amount will depend upon numerous factors, including, but notlimited to the components and physical characteristics of thetherapeutic composition, intended patient population, individual patientconsiderations, and the like, and can readily be determined by oneskilled in the art. “Pharmaceutically acceptable carrier” is a substancethat may be added to the active ingredient to help formulate orstabilize the preparation and causes no significant adversetoxicological effects to the patient. Examples of such carriers are wellknown to those skilled in the art and include water, sugars such asmaltose or sucrose, albumin, salts such as sodium chloride, etc. Othercarriers are described for example in Remington's PharmaceuticalSciences by E. W. Martin. Such compositions will contain atherapeutically effective amount of at least one anti-TFPI monoclonalantibody.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art. Thecomposition is preferably formulated for parenteral injection. Thecomposition can be formulated as a solution, microemulsion, liposome, orother ordered structure suitable to high drug concentration. The carriercan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Insome cases, it will include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, some methods of preparation are vacuumdrying and freeze-drying (lyophilization) that yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The human monoclonal antibody can be used for therapeutic purposes fortreating genetic and acquired deficiencies or defects in coagulation.For example, the human monoclonal antibodies may be used to block theinteraction of TFPI with FXa, or to prevent TFPI-dependent inhibition ofthe TF/FVIIa activity. Additionally, the human monoclonal antibody mayalso be used to restore the TF/FVIIa-driven generation of FXa to bypassthe insufficiency of FVIII- or FIX-dependent amplification of FXa.

The human monoclonal antibodies have therapeutic use in the treatment ofdisorders of hemostasis such as thrombocytopenia, platelet disorders andbleeding disorders (e.g., hemophilia A, hemophilia B and hemophlia C).Such disorders may be treated by administering a therapeuticallyeffective amount of the anti-TFPI monoclonal antibody variant to apatient in need thereof. The human monoclonal antibodies also havetherapeutic use in the treatment of uncontrolled bleeds in indicationssuch as trauma and hemorrhagic stroke. Thus, also provided is a methodfor shortening the bleeding time comprising administering atherapeutically effective amount of an anti-TFPI human monoclonalantibody variant of the invention to a patient in need thereof.

The antibodies can be used as monotherapy or in combination with othertherapies to address a hemostatic disorder. For example,co-administration of one or more variant antibodies of the inventionwith a clotting factor such as factor VIIa, factor VIII or factor IX isbelieved useful for treating hemophilia. In a separate embodiment,Factor VIII or Factor IX are administered in the substantial absence ofFactor VII. “Factor VII” includes factor VII and factor VIIa.

A method for treating genetic and acquired deficiencies or defects incoagulation comprises administering (a) a first amount of a variantmonoclonal antibody that binds to human tissue factor pathway inhibitorand (b) a second amount of factor VIII or factor IX, wherein said firstand second amounts together are effective for treating said deficienciesor defects. Similarly, a method for treating genetic and acquireddeficiencies or defects in coagulation comprises administering (a) afirst amount of a monoclonal antibody variant that binds to human tissuefactor pathway inhibitor and (b) a second amount of factor VIII orfactor IX, wherein said first and second amounts together are effectivefor treating said deficiencies or defects, and further wherein factorVII is not coadministered. The invention also includes a pharmaceuticalcomposition comprising a therapeutically effective amount of thecombination of a monoclonal antibody variant of the invention and factorVIII or factor IX, wherein the composition does not contain factor VII.

These combination therapies are likely to reduce the necessary infusionfrequency of the clotting factor. By co-administration or combinationtherapy is meant administration of the two therapeutic drugs eachformulated separately or formulated together in one composition, and,when formulated separately, administered either at approximately thesame time or at different times, but over the same therapeutic period.

In some embodiments, one or more antibody variants described herein canbe used in combination to address a hemostatic disorder. For example,co-administration of two or more of the antibody variants describedherein is believed useful for treating hemophilia or other hemostaticdisorder.

The pharmaceutical compositions may be parenterally administered tosubjects suffering from hemophilia A or B at a dosage and frequency thatmay vary with the severity of the bleeding episode or, in the case ofprophylactic therapy, may vary with the severity of the patient'sclotting deficiency.

The compositions may be administered to patients in need as a bolus orby continuous infusion. For example, a bolus administration of anantibody variant present as a Fab fragment may be in an amount of from0.0025 to 100 mg/kg body weight, 0.025 to 0.25 mg/kg, 0.010 to 0.10mg/kg or 0.10-0.50 mg/kg. For continuous infusion, an antibody variantpresent as an Fab fragment may be administered at 0.001 to 100 mg/kgbody weight/minute, 0.0125 to 1.25 mg/kg/min., 0.010 to 0.75 mg/kg/min.,0.010 to 1.0 mg/kg/min. or 0.10-0.50 mg/kg/min. for a period of 1-24hours, 1-12 hours, 2-12 hours, 6-12 hours, 2-8 hours, or 1-2 hours. Foradministration of an antibody variant present as a full-length antibody(with full constant regions), dosage amounts may be about 1-10 mg/kgbody weight, 2-8 mg/kg, or 5-6 mg/kg. Such full-length antibodies wouldtypically be administered by infusion extending for a period of thirtyminutes to three hours. The frequency of the administration would dependupon the severity of the condition. Frequency could range from threetimes per week to once every two weeks to six months.

Additionally, the compositions may be administered to patients viasubcutaneous injection. For example, a dose of 10 to 100 mg anti-TFPIantibody can be administered to patients via subcutaneous injectionweekly, biweekly or monthly.

Variant monoclonal antibodies to human tissue factor pathway inhibitor(TFPI) are provided. Further provided are the isolated nucleic acidmolecules encoding the same. Pharmaceutical compositions comprising thevariant anti-TFPI monoclonal antibodies and methods of treatment ofgenetic and acquired deficiencies or defects in coagulation such ashemophilia A and B are also provided. Also provided are methods forshortening the bleeding time by administering an anti-TFPI monoclonalantibody to a patient in need thereof. Methods for producing a variantmonoclonal antibody that binds human TFPI according to the presentdisclosure are also provided.

The therapeutic composition comprises antibody having binding regionsthat differ from the sequence of a parenteral TFPI binding antibody bythe intentional selection or engineering of one or more substitutions ofthe amino acid histidine (H, HIS) for at least one native amino acid asdefined in the parental sequence. The amino acid change confers longercirculating half-life T½ relative to the parental molecule.

An antibody specific for TFPI that binds to TFPI with at least 20% lowerefficiency at pH 6.0 than at pH 7.0 is disclosed and that shows animprovement in circulating T½ of approximately 400%. The beneficialeffect reducing TMDD is demonstrated for an antibody or antibody bindingregion that differs from the sequence of a target antibody such as2A8-g200 or 4B7-gB9.7 by the substitution of the amino acid histidine(H, HIS) for at least one native amino acid as defined relative to theparental sequence. Specifically, a variant of 2A8-g200 may have any oneof the following substitutions: VL-Y31H, VH-Y102H, VH-Y100H, VH-Y32H,VL-F48H, VL-S50H, VL-Y49H, VL-L27H, VL-V45H, VL-W90H and combinationsthereof.

Anti-TFPI antibody 2A8-g200 and 4B7-gB9.7 variants can bind to TFPI withhigh affinity and high specificity in vivo (see WO 2011/109452). FIG. 1shows amino acid sequence information for 2A8-g200 and 4B7-gB9.7, aswell as other 2A8 and 4B7 variants described in WO 2011/109452. Table 1shows the corresponding SEQ ID NOs for the variable heavy and variablelight chains for the 2A8 and 4B7 variants shown in FIG. 1.

TABLE 1 Corresponding SEQUENCE ID NOs of variable heavy and variablelight chains of the human anti-TFPI antibodies shown in Figure 1. CHAINMAb SEQ ID NO: VH 2A8 SEQ ID NO: 1 2A8-127 SEQ ID NO: 5 2A8-143 SEQ IDNO: 6 2A8-200 SEQ ID NO: 7 2A8-216 SEQ ID NO: 8 2A8-227 SEQ ID NO: 92A8-g200 SEQ ID NO: 10 2A8-g216 SEQ ID NO: 11 4B7 SEQ ID NO: 3 4B7-B18.5SEQ ID NO: 19 4B7-B2.0 SEQ ID NO: 20 4B7-B27.1 SEQ ID NO: 21 4B7-B32.5SEQ ID NO: 22 4B7-B41.2 SEQ ID NO: 23 4B7-B9.7 SEQ ID NO: 24 4B7-gB9.7SEQ ID NO: 25 4B7-gB9.7-IgG SEQ ID NO: 26 VL 2A8 SEQ ID NO: 2 2A8-127SEQ ID NO: 12 2A8-143 SEQ ID NO: 13 2A8-200 SEQ ID NO: 14 2A8-216 SEQ IDNO: 15 2A8-227 SEQ ID NO: 16 2A8-g200 SEQ ID NO: 17 2A8-g216 SEQ ID NO:18 4B7 SEQ ID NO: 4 4B7-B18.5 SEQ ID NO: 27 4B7-B2.0 SEQ ID NO: 284B7-B27.1 SEQ ID NO: 29 4B7-B32.5 SEQ ID NO: 30 4B7-B41.2 SEQ ID NO: 314B7-B9.7 SEQ ID NO: 32 4B7-gB9.7 SEQ ID NO: 33 4B7-gB9.7-IgG SEQ ID NO:34

pH sensitive variants of 2A8-g200 and 4B7-gB9.7 were created bysubjecting both the CDR domains and the residues contacting the TFPI toanalysis for binding characteristics upon mutagenesis at selected sites.FIG. 2 shows the location of possible His mutations for: A. 2A8-g200(designated as A200 in FIG. 2A) variable heavy chain; B. 2A8-g200(designated as A200 in FIG. 2B) variable light chain; C. 4B7-gB9.7variable heavy chain; and D. 4B7-gB9.7 variable light chain. Onehistidine residue was substituted for each of the amino acids ineither 1) a contact residue to TFPI as indicated by an underlined aminoacid in FIG. 2, or 2) a CDR 1-3 residue as indicated by an asterisk inFIG. 2 for the anti-TFPI antibodies 2A8-g200 and 4B7-gB9.7. As shown inFIGS. 2A and 2B, forty (40) residues from the heavy chain andtwenty-nine (29) residues from the light chain were identified as thepositions for mutagenesis in 2A8-g200. As shown in FIGS. 2C and 2D,forty (40) heavy chain and thirty-two (32) light chain variants wereidentified in 4B7-gB9.7.

A 2A8-g200 Fab histidine scanning library was synthesized. The librarycontained 69 members. The 2A8-g200 Fab histidine library was cloned intoa bacterial expression vector and the amino acid sequences wereverified.

Sixty-nine (69) clones from the His scan library were transformed intoE. coli ATCC strain 9637 and grown on selective media containingcarbenecillin (100 μg/mL). Single colonies were used to inoculateLB-Carbenecillin-100 media. The cultures were grown to OD600=0.5 at 37°C., induced with 0.25 mM IPTG, and grown overnight at 30° C. Thebacterial expression cultures were harvested by centrifugation at5,000×g for 15 min at 4° C. The expression media was decanted from thepellet. Both pellet and cleared expression media were frozen at −20° C.The His muteins were purified from the expression media with Protein A.Purified muteins were analysed by SDS-PAGE and a concentration wasobtained by A280.

Human TFPI, 1 μg/ml, was used to coat Maxisorb™ microtiter plates.Expression media, 100 μl, from each member of the His scan library, wasadded to two wells on the plate, in a pair wise fashion. The plate wasincubated on a shaker at room temperature for 1 hr. The plate was washed3× with PBST. PBS (pH 7.0) was added to one well of the pair, 100 mMpH6.0 Citrate buffer was added to the second well of the same pair. Theplate was incubated at 37° C. with shaking for one hr. The plate waswashed 3× with PBST and developed using amplex red. A pH 7.0/pH 6.0ratio was established to rank the sensitive muteins. The ratio for wildtype 2A8-g200 Fab was 1.0. The 10 clones that had a ratio greater than1.78 between pH 7.0 and pH 6.0 are shown in Table 2 below.

TABLE 2 pH 6.0 TFPI Dissociation ELISA TFPI ELISA Ratio Rank Mutation pH7.0 pH 6.0 pH 7.0/pH 6.0 wt gA200Fab 5248 5354 0.98 1 VL-Y31H 913 1336.64 2 VH-Y102H 3310 1079 3.07 3 VH-Y100H 2545 1431 1.78 4 VH-Y32H 35602585 1.38 5 VL-F48H 2068 1551 1.33 6 VH-S50H 2159 1637 1.32 7 VL-Y49H2661 2044 1.3 8 VL-L27H 3422 2637 1.3 9 VL-V45H 2197 1771 1.24 10VL-W90H 1833 1509 1.21

Purified 2A8-g200 variants in Fab format (referred to as wt gA200Fab inTable 1) were tested using surface plasmon resonance (Biacore). Surfaceplasmon resonance (Biacore T200) was used to measure the dissociationrate of the antibodies. Human TFPI (American Diagnostica) was aminecoupled on a CM4 or CM5 chip using the method suggested by Biacore,resulting in 100 to 300 RU of immobilized TFPI. Purified 2A8-g200variants were injected, following by 40-minute dissociation either atpH7.4 or pH6.0 buffer. The antibodies were diluted in HBS-P buffer atdifferent concentrations and the flow rate was set to 50 μl/min. Aftereach round of antibody injection, the chip was regenerated by injecting90 μl of pH 1.5 glycine. The data set was evaluated using BIAevaluationSoftware.

The dissociation constant (kd) for each 2A8-g200 variant antibody wasdetermined by using a model with the following equation:R=R ₀ e ^(−kd(t−t) ⁰ ⁾+offsetwhere R is the response at time t, R₀ is the response at time t₀.—thestart of dissociation, offset allows for a residual response at the endof complete dissociation. A ratio of kd at pH 6.0 to kd at pH 7.4 wascalculated for each 2A8-g200 variant. A mutation with observed ratio of2 was considered as pH-sensitive mutation and could be used forconstruction of IgG variants of 2A8-g200.

For example, referring to FIG. 3, observed variations in dissociationconstant responses in the biocore assay at two different pHs (pH 6.0 andpH 7.4) are shown for two exemplary 2A8-g100 light chain histidinesubstitution mutations: A. L-L27H and B. L-Y31H.

Pharmacokinetic parameters of the antibodies were determined afterintravenous (i.v.) bolus administration to HemA mouse at 2 mg/kg. Allthe pharmacokinetic parameters were calculated using WinNonLin softwareversion 5.3.1 (Pharsight Corporation, Mountain View, Calif.)non-compartmental model. The effect of histidine mutations on theobserved half-life of anti-TFPI monoclonal antibodies in mouse plasma isshown in FIG. 4. 2A8-g200 with the histidine mutations TPP2256(L-Y31H/Y49H) and TPP2259 (L-Y31H) increased the observed pK profilesover a 500 hour time span as compared to the corresponding pK profile of2A8-g200 without any histidine substitution. Table 3 quantifies theincreases in half-life observed in the data of FIG. 4.

TABLE 3 pK parameters Dose T ½ Antibody (mg/kg) (hr) 2A8-g200 2 71TTP-2256 2 ~331 TTP-2259 2 ~437

Therefore, the above designated antibodies that reduce TFPI mediatedTMDD and have a prolonged T½ would lead to less frequent dosing andreduce the amount of material needed per dose. Furthermore, the need fora lower dose may also make feasible subcutaneous dosing where the dosingvolume becomes a limiting step, a process by which the antibody isremoved from circulation due to its interaction with a rapidly clearedtarget or by being sequestered from the plasma due to itsco-localization with its target.

There will be various modifications, improvements, and applications ofthe disclosed invention that will be apparent to those of skill in theart, and the present application encompasses such embodiments to theextent allowed by law. Although the present invention has been describedin the context of certain preferred embodiments, the full scope of theinvention is not so limited, but is in accord with the scope of thefollowing claims. All references, patents, or other publications arespecifically incorporated by reference herein.

The invention claimed is:
 1. A variant of human monoclonal IgG antibodythat binds specifically to human tissue factor pathway inhibitor (TFPI),which comprises a variable light chain domain and a variable heavy chaindomain, and has increased plasma half-life as compared to the parentalhuman monoclonal IgG antibody, wherein the variant antibody furthercomprises: a variable heavy chain domain defined by parental SEQ ID NO:17, which comprises a histidine substitution selected from the groupconsisting of Y102H, Y32H and Y100H, and combinations thereof; and thevariant antibody binds to TFPI at pH 7.0 with at least two fold higheraffinity than at pH 6.0.
 2. A variant of human monoclonal IgG antibodythat binds specifically to human tissue factor pathway inhibitor (TFPI),which comprises a variable light chain domain and a variable heavy chaindomain, and has increased plasma half-life as compared to the parentalhuman monoclonal IgG antibody, wherein the variant antibody furthercomprises: a variable light chain domain defined by parental SEQ. ID.NO. 10, which comprises a histidine substitution selected from the groupconsisting of Y31H, F48H, Y49H, L27H, V45H, W90H and combinationsthereof; and the variant antibody binds to TFPI at pH 7.0 with at leasttwo fold higher affinity than at pH 6.0.
 3. The variant antibody ofclaim 1, further comprising the variable light chain domain of SEQ IDNO:
 10. 4. The variant antibody of claim 1, further comprising avariable light chain domain defined by parental SEQ. ID. NO. 10, whichcomprises a histidine substitution selected from the group consisting ofY31H, F48H, Y49H, L27H, V45H, W90H, and combinations thereof.
 5. Thevariant of claim 2, further comprising the variable heavy chain domainof SEQ ID NO:
 17. 6. A pharmaceutical composition comprising the variantantibody of claims 1, 2, 3, 4, or 5 and a pharmaceutically acceptablecarrier.